To understand the temporal gene expression profile of IDGF1, IDGF3_1, IDGF4_0, IDGF4_1, and IDGF6 of Z. cucurbitae, RT-qPCR was performed at different developmental stages. RT-qPCR was performed using SYBR® Premix Ex TaqTM II (TliRNaseH Plus) (Takara, Japan) on an ABI 7500 instrument (United States). The PCR reaction includes 10 μl SYBER Green mix, 1 μl cDNA, 1 μl each of forward and reverse primers and 7 μl of ddH2O with three technical and three biological replicates for each gene expression. The elongation factor 1 alpha (EF1α) was used as endogenous reference genes for data normalization, and a relative transcript level of IDGFs was calculated with the 2–ΔΔCt method (Livak and Schmittgen, 2001). All the primers used in this study are shown in Supplementary Table 3.

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